The major physiologically active metabolite of vitamin A, retinoic acid (RA), is important in preventing certain types of cancer. RA can also arrest the growth and induce differentiation of a variety of tumor cells in culture and is used to treat certain cancers. We have been studying RA-induced melanoma differentiation. An important finding has been the key role played by PKCalpha in the differentiation pathway. We will continue studies on the involvement of PKC and PKC-mediated events in melanoma differentiation. Specifically, we will determine whether RARbeta is directly involved in the induction of PKCalpha and subsequent differentiation by: establishing stable transfectants in which the expression of RARbeta is under the control of an inducible promoter; disrupting the RARbeta gene through homologous recombination; use of new retinoid analogs which have specificity for activating RARbeta. By combining the data from three separate approaches we should obtain convincing evidence regarding the role of RARbeta in melanoma differentiation. We found that the RA also induces an increase in AP-1 activity. This is not due to an increase in c-fox or c-jun nor to an increase in binding to an AP-1 element, but apparently to an increase in transcriptional activity of c-jun amino-terminal kinase (JNK), or increasing its expression. In order to determine the importance of AP-1 activity for differentiation we will isolate stable transfectants which express a dominant-negative mutant of c-jun and test whether these clones are refractory to RA. We will also use newly developed retinoid analogs which distinguish between transactivation and AP-1 transrepression of RAR. An exciting finding of the previous grant period was that PKC could positively regulate RAR function, thus creating a positive feed-back loop. We will investigate whether PKCalpha can directly phosphorylate the RARs and how this phosphorylation affects receptor functions. These data will provide further insight into the process of RA-mediated tumor cell differentiation and could suggest means by which the process could be potentiated.